We will address PQ4 ? the need to develop tools for simultaneous manipulation of multiple genes in human cancer-relevant models ? because a key challenge in the field is to understand how multiple genes affected by epigenetic or copy number alterations act in concert to influence the process of tumor development. For the epigenetically affected genes, we have shown that multiple important tumor suppressor and developmental regulator genes are maintained in a permanently silenced state by promoter CpG-island (CGI) DNA hypermethylation across various cancer types. Importantly many genes from the same biological pathways are epigenetically silenced within the same patient sample. The cumulative effect of multiple epigenetically silenced genes (ESGs) on tumor development remains an unaddressed aspect of cancer biology, mainly because of a lack of tools for targeting multiple genes, tractable biological models and assays to measure tumor development. In this proposal we seek to fill these gaps by leveraging our current work in human cancer relevant models. Our motivation to pursue these studies originate from our work in the past 3-4 years where we have modeled the early stages of human colorectal cancer (CRC) development by oncogenic BRAFV600E and KRASG12D mutations. We show that in mouse proximal colon-derived organoids, induction of BrafV600E, but not mutant KrasG12D, results in Wnt pathway activation, stem cell phenotype and transformation. Tumor explants of the transformed organoids phenocopy human proximal CRC, inclusive of mucinous histopathology and CGI hypermethylator phenotype (CIMP). We show that an aging-like acquisition of CGI hypermethylation in organoids, not unlike that in aging human colon, promotes BrafV600E-mediated tumorigenesis by inducing early Wnt pathway activation and stem cell phenotype, which are early features during tumorigenesis in this model. Our studies, in concert with work from other labs, show that the organoids are a highly relevant model that phenocopies features of human tumor progression. Further, we have established Cas9 and Cpf1-based screening in organoids to identify loss of ESGs that promote tumorigenesis. The major goal of this project is to develop tools for simultaneous inactivation of multiple ESGs to study their combined roles in cancers. In this regard, we have developed inducible-CRISPR based human colon-derived organoids for multiple gene knockout to study the collusion between ESGs and cancer driver mutations. We will continue on these by developing human and mouse colon organoid system with inducible Cas9/Cpf1 for systematically screening random combinatorial inactivation of multiple ESGs that confer stem-cell maintenance and block differentiation. Next we will focus on important candidate genes, bioinformatically mined form cancer databases, to study the synergy of their simultaneous loss in cancer initiation. Finally, we will apply and validate our studies in in-vivo mouse models. In summary, this proposal will develop generic tools for simultaneous manipulation of multiple genes in human cancer-relevant models that can be easily translated to other cancer models.